The β-propeller domain of HCF-1 is sufficient for interaction with Bap1. (A) Schematic representation of HCF-1 to indicate the positions of the β-propeller, basic region, HCF_PRO repeats (arrowheads), transactivation domain (AD), and tandem Fn3 repeats. Four HCF-1 truncations used in panel B have been described previously (43) and correspond to HCF-1Δrep (NC), HCF-1_N1011 (N), HCF-1_N380 (N_β), and HCF-1_N450-1011 (N_basic). An HA-epitope tag is included at the N terminus of each HCF-1 polypeptide. (B) The HCF-1 β-propeller domain is sufficient for interaction with Bap1. Plasmids encoding Bap1-Flag and selected HA-tagged HCF-1 truncations were transiently transfected into 293 cells, and their association was examined by coimmunoprecipitation. Cells were lysed in 1% digitonin and Bap1-Flag immunoprecipitated from the clarified lysates using anti-Flag beads. Recovered proteins were detected by immunoblotting anti-HA and anti-Flag antibodies. (C) Catalytic activity of Bap1 is not required for association with endogenous HCF-1. Control 293 cells (mock) or cells stably expressing low levels of wild-type (Wt) or active site mutant (C91A) Bap1-Flag were lysed in 1% digitonin and immunoprecipitated using anti-Flag beads. Endogenous HCF-1 was detected by immunoblotting with anti-HCF-1 antibody.

Endogenous HCF-1 is ubiquitinated, and the major site of HCF-1 ubiquitination is mapped to K1807 or K1808 residues. (A) Scheme for partial purification of ubiquitinated HCF-1 through association with Bap1-Flag. (B) Samples from Bap1-Flag-expressing or control 293 cells were subjected to sequential anti-Flag and anti-HA immunoprecipitation as summarized for panel A and analyzed by MS/MS. Peptide masses and fragment ions characteristic of two tryptic peptides derived from HCF-1_C were monitored throughout an LC-MS/MS experiment. Three fragment ions for each peptide were detected in the samples from Bap1-Flag-expressing cells but not the controls. (C) Purification of HCF-1-V5 for ubiquitination site mapping. HA-Ub was expressed in 293 cells with or without HCF-1-V5. HCF-1-V5 polypeptides were recovered from denatured lysates by immunoprecipitation with anti-V5 beads. After extensive washing, bound proteins were eluted and detected by immunoblotting with anti-V5 and anti-HA antibodies (left panel) or directly visualized by silver staining (right panel). (D) Identification of lysine-1807 and/or lysine-1808 of HCF-1_C as major sites of ubiquitination. Eluted HCF-1-V5 and control samples (from C) were analyzed by MS/MS, and a single HCF-1 peptide was identified to be ubiquitinated. Highlighted fragment ions are derived from HCF-1_C peptide 1804-APMKKENQWFDVGVIK-1809 and display a mass shift indicative of a ubiquitin linkage at lysine-1807 and/or lysine-1808.

Catalytically inactive Bap1 promotes the selective accumulation of K48 ubiquitinated HCF-1. (A) HCF-1 is ubiquitinated through both lysine-48 (K48) and lysine-63 (K63) linkages. 293 cells were transiently transfected with plasmid encoding HCF-1-V5 and HA-tagged K48 or K63-ubiquitin. Cells were treated for 2 h with 20 μM MG132 prior to lysis in NP-40-containing buffer supplemented with 1% SDS. After removal of the insoluble material, the lysates were diluted in 10 volumes of buffer lacking SDS, and V5-tagged HCF-1 was recovered by anti-V5 immunoprecipitation. Linkage of ubiquitin moieties was detected by immunoblotting with anti-HA antibody. (B) Ectopic expression of Bap1 alters the level of HCF-1 K48 ubiquitination. Plasmids encoding HCF-1-V5, K48 HA-Ub, and wild-type or C91A mutant Bap1-Flag were introduced into 293 cells and processed as described for panel A. (C) Bap1 expression only affects levels of K48 ubiquitinated HCF-1. HCF-1-V5, C91A Bap1-Flag, K48 HA-Ub, or K63 HA-Ub were expressed as indicated in 293 cells. Prior to lysis, cells were treated for 2 h with 20 μM MG132. HCF-1-V5 was immunoprecipitated (NP-40 + 1% SDS lysis, followed by 10-fold dilution in buffer without SDS) with anti-V5 antibody, and equal amounts of HCF-1-V5 were loaded on gels, followed by Western blotting with anti-HA antibody. The results show that levels of K48 ubiquitinated HCF-1 are considerably increased when C91A Bap1 is expressed; however, no noticeable effects on the levels of K63 ubiquitinated HCF-1 were observed.

HCF-1 associates with Bap1 in transfected cells. (A) Sequences of 27 HCF-1-derived peptides identified by MS/MS from proteins coimmunoprecipitated with Bap1-Flag. The amino acid positions in the prototype human HCF-1 protein sequence (RefSeq accession no. NP_005325) are indicated. (B) Coassociation of Bap1-Flag and HCF-1-V5 in transfected cells. Bap1-Flag and HCF-1-V5 were expressed individually or together in human 293 cells, and their expression was confirmed in immunoblotting of the 1% digitonin lysates (left panels). Bap1-Flag was immunoprecipitated using Flag antibody and precipitated proteins detected by immunoblotting with V5 or Flag (right panels). (C) Bap1 and HCF-1 colocalize within the cell. HeLa cells were transiently transfected with a Bap1-HA expression plasmid, fixed and subjected to indirect immunofluorescence using an α-HA monoclonal antibody to detect Bap1 and anti-HCF-1 (N18) polyclonal antibody to detect endogenous HCF-1. Scale bar, 5 μm. (D) Bap1-Flag associates with endogenous HCF-1 both in cytoplasm and nucleus. Whole-cell lysates (T) or cytoplasmic (C) and nuclear (N) fractions were prepared from control 293 cells or cells stably expressing low levels of Bap1-Flag and probed by immunoblotting either directly or after immunoprecipitation with anti-Flag beads. Sp1 serves as a nuclear protein control.

Identification of HCF-1 as a Bap1 interacting protein. (A) Schematic comparing structures of human UCH family members: Bap1 (RefSeq accession no. NP_004647), UCH37 (NP_057068), UCHL1 (NP_004172), and UCHL3 (CAG33136). The Bap1 UCH domain is most similar to that of UCH37 (42.5% sequence identity) and includes the critical cysteine, histidine, and aspartate residues of the active site. Bap1 and UCH37 share an additional region of homology at the C terminus (ULD, green box). In UCH37, this region folds as a coiled coil that interacts with the Rpn13 subunit of the proteasome. (B) Sequences of the C-terminal homology region (ULD) from human and mouse Bap1 (accession numbers NP_004647 and NP_081364) and UCH37 (accession numbers NP_057068 and AAD31534). Residues in UCH37 that are identical (filled) or similar (shaded) to Bap1 are indicated. (C) Purification of Flag-tagged Bap1 (Bap1-Flag). Silver-stained SDS-PAGE gel (4 to 12% gradient) showing eluted proteins after anti-Flag coimmunoprecipitation from a 1% digitonin lysate of Bap1-Flag-expressing 293 cells. Bands from a Bap1-Flag sample and corresponding regions of the control sample were excised, digested with trypsin, and subjected to peptide analysis by MS/MS. In three independent experiments, multiple bands in the 100-kDa or larger size range were identified as HCF-1 from the Bap-1 elution but not the control.

Bap1 contributes to HCF-1 stability. (A) Depletion of Bap1 using siRNA. Control 293 cells or cells stably expressing Bap1-Flag were treated with control or Bap1 siRNA for 3 days. Endogenous and ectopic Bap1 were immunoprecipitated and blotted with a rabbit polyclonal antibody 419 against Bap1 (α-Bap1). (B) Depletion of Bap1 leads to a modest stabilization of HCF-1. 293 cells were treated with control siRNA or Bap1 siRNA for 2 days and then transfected with HCF-1-V5. After 48 h, cells were lysed, and the clarified lysates were probed by immunoblotting with anti-V5 antibody. Blotting for tubulin served as a loading control. (C) Depletion of Bap1 alters cell cycle progression. Subconfluent HeLa cells were cotransfected with a GFP expression plasmid and either a Bap1 siRNA or a control siRNA. After 48 h, cells were fixed and stained with propidium iodide, and GFP-positive cells were analyzed for DNA content by flow cytometry. For easier comparison of the two FACScalibur profiles, a dotted line marks the 4N peak in the mock siRNA sample. (D) Schematic showing the proposed association of Bap1 with the two subunits of HCF-1. Bap1 contains a nonconsensus HBM that is recognized by the β-propeller domain of the HCF-1_N subunit and required for association of Bap1 with HCF-1. By analogy to UCH37, the C-terminal coiled-coiled (C-C) region of Bap1 may facilitate recruitment to the proteasome, whereas the UCH domain catalyzes the removal of K48-linked ubiquitin moieties. A candidate substrate might be the HCF-1_C subunit, which is shown to be ubiquitinated at conserved lysine-1808 and/or lysine-1809 in the first Fn3 repeat.

Association with HCF-1 requires a conserved tetrapeptide HBM in the central region of Bap1. (A) An HBM-like sequence is located near the center of Bap1, within a region that is highly conserved in vertebrate Bap1 sequences. Accession numbers are as follows: human (Homo sapiens), NP_004647; mouse (Mus musculus), NP_081364; chicken (Gallus gallus), NP_001025761; zebrafish (Danio rerio), XP_687254; and African clawed toad (Xenopus laevis), NP_001089388. (B) Mutation of the NHNY sequence abrogates the interaction with HCF-1. Empty Flag vector (lanes 1 and 5) or vectors encoding wild-type Bap1-Flag (lanes 2 and 6), C91A Bap1-Flag (lanes 2 and 6), or NHNY/AAAA Bap1-Flag (lanes 4 and 8) were transiently cotransfected into 293 cells, together with either empty Myc tag vector (lanes 1 to 4) or HCF1-Myc vector (lanes 5 to 8). Cells were lysed in buffer containing 1% digitonin, immunoprecipitated with anti-Flag antibody beads, and coprecipitated HCF-1-Myc detected by immunoblotting with anti-Myc antibody. (C) The NHNY/AAAA Bap1 mutant retains enzymatic activity. Immunopurified Flag-tagged proteins were washed extensively, eluted from the beads with free three-Flag peptide, and assayed for the ability to cleave a ubiquitin-AMC fluorogenic substrate. Purified UCH-L3 and cells transfected with empty vector served as positive and negative controls. Values correspond to the average of three independent assays, and the standard deviations are shown.